Antibiotic resistance has recently been identified as one of the three greatest threats facing mankind in the 21st century by World Health Organization. One promising approach to combat antibiotic resistance is to reinvesti- gate known antibiotics and design their derivatives, in the hope of identifying novel antibiotic agents that com- bat antibiotic resistance. Hydantoins, the derivatives of 2,4-imidazolidinedione, have been developed for anti- bacterial applications for long time. The mechanism of action for hydantoin derivatives is complex and not well understood, possibly due to their damage to bacterial DNA, as well as bacterial ribosome binding and inhibition of critical bacterial enzymes. One hydantoin derivative, nitrofurantoin, was approved to treat urinary tract infec- tions. As an old antibiotic, it recently attracted considerable interest due to their low probability of bacterial re- sistance compared to other conventional antibiotics such as fluoroquinolones, possibly owing to their mixed mechanism of action. However, hydantoin derivatives including nitrofurantoin generally exhibit only moderate antibacterial activity, which limits their further application in combating emergent antibiotic resistance. In the last RO1 period, we have developed a series of novel antimicrobial AApeptides. Through proper de- sign and modification, we have recently developed a series of novel membrane-active hydantoin derivatives derived from AApeptides that display potent and broad-spectrum antimicrobial activity (25-100 fold of nitrofu- rantoin) in vitro and in vivo. Our preliminary studies strongly suggest these compounds as a new approach for antibiotic development. As such, our long-term goal is to develop novel antibiotic agents with novel mecha- nisms to combat drug-resistant bacterial infections. The objective here, is to further develop these hydantoin derivatives with greater potency through optimization of current lead compounds. Our central hypothesis is that these agents, with proper design and modification, could be further improved in bacterial killing through novel mechanisms. To test our central hypothesis and, thereby, accomplish the objective of this application, we will first design and synthesize analogs of previously developed lead molecules, and identify more potent mole- cules that are active against both Gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) (MIC ? 0.5 g/ml) and Gram-negative Pseudomonas aeruginosa (MIC ? 1 g/mL). Next, we will study if bactericidal mechanism of lead compounds involves membrane action, and assess their probability to elicit antibiotic re- sistance. Furthermore, we will evaluate the in vivo activity of lead compounds in a thigh-infection mouse model, in order to demonstrate their potential as a new generation of antibiotics with novel mechanisms. The work proposed is innovative because these compounds are a new class of hydantoin compounds that kill both Gram-positive and Gram-negative bacteria with novel mechanisms. They are highly amendable for derivatization and optimization, and possess low propensity to induce antibiotic resistance. The proposed work is significant because currently there are no effective methods to combat emerging drug resistance. Our re- search strategy will lead to a promising therapeutic approach to treat antibiotic resistant pathogens.